* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download article in press - MRC
Epigenetics of diabetes Type 2 wikipedia , lookup
Genomic library wikipedia , lookup
Extrachromosomal DNA wikipedia , lookup
Neuronal ceroid lipofuscinosis wikipedia , lookup
Cancer epigenetics wikipedia , lookup
Population genetics wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Epigenomics wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Genome (book) wikipedia , lookup
Gene therapy wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
Saethre–Chotzen syndrome wikipedia , lookup
Genetic engineering wikipedia , lookup
SNP genotyping wikipedia , lookup
Non-coding DNA wikipedia , lookup
Genome evolution wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Epigenetics of neurodegenerative diseases wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
Pharmacogenomics wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Genome editing wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Cell-free fetal DNA wikipedia , lookup
History of genetic engineering wikipedia , lookup
Designer baby wikipedia , lookup
Microsatellite wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Oncogenomics wikipedia , lookup
Helitron (biology) wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Frameshift mutation wikipedia , lookup
ATH-9692; ARTICLE IN PRESS No. of Pages 10 Atherosclerosis xxx (2006) xxx–xxx Genetic defects causing familial hypercholesterolaemia: Identification of deletions and duplications in the LDL-receptor gene and summary of all mutations found in patients attending the Hammersmith Hospital Lipid Clinic Isabella Tosi a,1 , Paola Toledo-Leiva a,1 , Clare Neuwirth a,b , Rossi P. Naoumova a,b , Anne K. Soutar a,∗ b a MRC Clinical Sciences Centre, Imperial College London, United Kindom Lipid Clinic, Hammersmith Hospital, Du Cane Road, London W12 0NN, United Kingdom Received 3 July 2006; received in revised form 2 October 2006; accepted 6 October 2006 Abstract Familial hypercholesterolaemia (FH) results from defective catabolism of low density lipoproteins (LDL), leading to premature atherosclerosis and early coronary heart disease. It is commonly caused by mutations in LDLR, encoding the LDL receptor that mediates hepatic uptake of LDL, or in APOB, encoding its major ligand. More rarely, dominant mutations in PCSK9 or recessive mutations in LDLRAP1 (ARH) cause FH, gene defects that also affect the LDL-receptor pathway. We have used multiplex ligation-dependent probe amplification (MLPA) to identify deletions and rearrangements in LDLR, some not detectable by Southern blotting, thus completing our screening for mutations causing FH in a group of FH patients referred to a Lipid Clinic in London. To summarise, mutations in LDLR were found in 153 unrelated heterozygous FH patients and 24 homozygotes/compound heterozygotes, and in over 200 relatives of 80 index patients. LDLR mutations included 85 different point mutations (7 not previously described) and 13 different large rearrangements. The APOB R3500Q mutation was present in 14 heterozygous patients and a mutation in PCSK9 in another 4; LDLRAP1 mutations were found in 4 “homozygous” FH patients. Our data confirm that DNA-based diagnosis provides information that is important for management of FH in a considerable number of families. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Plasma cholesterol; MLPA; LDLRAP1; PCSK9; APOB; Heterozygous FH; Homozygous FH 1. Introduction Familial hypercholesterolaemia (FH) is an autosomal dominant disorder caused by defective clearance of low density lipoproteins (LDL) from the circulation, leading to premature atherosclerosis and a marked increased risk of coronary heart disease. FH is usually caused by mutations in the gene for the LDL receptor (LDLR); a mutation in the gene for the ligand for the LDL receptor, apolipoprotein B100 (ApoB100), results in the same phenotype, but this disorder ∗ 1 Corresponding author. Tel.: +44 20 8383 2324; fax: +44 20 8383 2028. E-mail address: [email protected] (A.K. Soutar). These authors contributed equally to this work. is often referred to as familial defective ApoB100 (FDB) [1]. During various studies in which we have investigated the genetic defect underlying a firm clinical diagnosis of definite heterozygous FH in patients in the UK, we have failed to detect a mutation in the genes for the LDL receptor (LDLR) or apolipoprotein B (APOB) in up to 15% of any group. At first, this was ascribed to lack of sensitivity in the analysis methods used, but the advent of high quality, automated nucleotide sequencing of PCR-amplified DNA has not greatly reduced the number of patients with an unidentified mutation, suggesting that some defects in the LDLR remain undetected or that other genes may be involved. The identification of dominant mutations in PCSK9 as a cause of heterozygous FH 0021-9150/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2006.10.003 Please cite this article in press as: Tosi I et al., Genetic defects causing familial hypercholesterolaemia: Identification of deletions and duplications in the LDL-receptor gene and summary of all mutations found in patients attending the Hammersmith Hospital Lipid Clinic, Atherosclerosis (2006), doi:10.1016/j.atherosclerosis.2006.10.003 ATH-9692; No. of Pages 10 2 ARTICLE IN PRESS I. Tosi et al. / Atherosclerosis xxx (2006) xxx–xxx have revealed that defects in other genes can result in this phenotype, but these remain a rare cause of FH [2]. In order to define a group of patients that warrant further investigation of the underlying cause of their inherited hypercholesterolaemia, it was necessary to exclude those with a large deletion or duplication in the LDLR. Although Southern blotting has been used successfully for this purpose by ourselves and others [3–7] it is time-consuming, technically demanding, and requires large amounts of high quality DNA. Most importantly, it lacks the sensitivity to detect all rearrangements, for example deletions encompassing most of the coding region of the gene so that a cDNA probe cannot readily detect abnormal restriction enzyme fragments. Recently, a novel method for the detection of gene deletions and duplications has been devised: multiplex ligationdependent probe amplification (MLPA) [8]. The method depends on the hybridisation of two short specific oligonucleotide probes to abutting regions of an exon in genomic DNA from a patient. If the exon is intact, the two oligonucleotides can be ligated and the resultant fragment amplified by PCR with fluorescently labelled primers for which sequences are incorporated at the end of each probe. Pairs of probes for many exons of one or more genes, all containing this same pair of universal primer sequences, can be hybridised, ligated and amplified in single multiplex reactions. The size of each amplified product is determined by the inclusion of a “filler” sequence of different length in each probe and thus the PCR products can readily be analysed on a DNA sequencer. Comparison of the relative amount of each product for the gene in question with that from control genes on different chromosomes allows the identification of exons that are absent (homozygous deletion) or present at half copy number (heterozygous deletion) or in multiple copy number (heterozygous duplication) in a patient. Here we describe its use to detect 12 different gene rearrangements in the LDLR, several of which would not be detected readily by Southern blotting. The majority of these have been confirmed by PCRamplification across the deletion/duplication joint in DNA or mRNA and/or by their presence in affected relatives. characterised by extreme hypercholesterolaemia with serum cholesterol between 14 and 30 mmol/l, onset of cutaneous planar or tuberous xanthomas in early childhood plus tendon xanthomas and corneal arcus. The patients were either referred to the Lipid Clinic or their samples were sent to us for molecular diagnosis. All patients had given informed consent to DNA-based diagnosis of their disorder. 2.2. Identification of point mutations Methods used to identify point mutations and minor insertion deletions in LDLR, APOB and PCSK9 by sequencing of amplified fragments of genomic DNA or mRNA from immortalized lymphocytes have been described in detail elsewhere [10–12]. 2.3. MLPA DNA samples from patients were analysed by MLPA according to the supplier’s instructions [13] and compared with at least five control samples analysed at the same time. The PCR products were fractionated on an ABI DNA sequencer and the data analysed with GenotyperTM software. Only electropherograms that passed quality control were analysed, i.e. the peak heights of non-ligated probes were negligible compared with ligated probe fragments. Peaks on each electropherogram were normalised by expressing their height as a fraction of the total height of all control peaks. Relative amounts of each LDLR exon in each patient sample were determined by dividing the normalised value for each peak by the average normalised value for that amplicon from at least five controls (variance <10%). Exons present at less than 0.75-fold the control value were deemed to have half the normal copy number, and those at more than 1.3-fold to be duplicated. Amplification of genomic DNA to confirm the presence of deletions was carried as described previously [10]; details of the primers and the conditions used are shown in Table 1. 3. Results 2. Methods 2.1. Patients The heterozygous FH patients whose DNA was analysed in this study had all attended the Hammersmith Hospital Lipid Clinic during the last 10–15 years. The diagnosis of FH was based on clinical criteria established by the Simon Broome Study Group [9], i.e. total plasma cholesterol concentration greater than 7.5 mmol/l in the proband, together with either tendon xanthoma in the proband or in a first degree relative (definite FH), or the presence of premature coronary heart disease or hypercholesterolaemia in a first degree relative (possible FH). The homozygous FH patients were Table 2 shows the different mutations that have been identified in 145 apparently unrelated heterozygous FH probands attending the Hammersmith Hospital Lipid Clinic over the last 10–15 years, while Table 3 summarises the genetic defects found in 28 patients with a clinical diagnosis of suspected homozygous FH who were either referred to the clinic or whose samples were sent to us for genetic characterisation of the underlying defect ([6,10,14–22] and unpublished data). Several additional mutations have also been identified by us in a group of heterozygous FH patients from Cardiff [10], and in both homozygous and heterozygous patients of Chinese origin [23,24]. Patients in whom no defect could be identified by sequencing were analysed for major deletions and insertions in Please cite this article in press as: Tosi I et al., Genetic defects causing familial hypercholesterolaemia: Identification of deletions and duplications in the LDL-receptor gene and summary of all mutations found in patients attending the Hammersmith Hospital Lipid Clinic, Atherosclerosis (2006), doi:10.1016/j.atherosclerosis.2006.10.003 ATH-9692; No. of Pages 10 ARTICLE IN PRESS I. Tosi et al. / Atherosclerosis xxx (2006) xxx–xxx 3 Table 1 PCR primers and conditions for confirmation of rearrangements in LDLR LDLR mutation Primers: name Sequence Conditions for mutant allele Del e5 F: AKS54; R: 6R 5 -CCCCAGCTGTGGGCCTGCGACA-3 ; Del e13–14 F: AKS230; R: AKS233 Del e16–17 F: 15F; R: 18R Dup e9–14 F: AKS232; R: AKS227 Dup e11–12 F: 12F; R: 11R 94 ◦ C × 2 ; 94 ◦ C × 30 , 62 ◦ C × 45 , 68 ◦ C × 1 /30 cycles; 68 ◦ C × 8 94 ◦ C × 2 ; 94 ◦ C × 1 , 65 ◦ C × 3 , 68 ◦ C × 1 /35 cycles; 68 ◦ C × 8 94 ◦ C × 2 ; 94 ◦ C × 30 , 59 ◦ C × 45 , 68 ◦ C × 4 /35 cycles; 68 ◦ C × 8 94 ◦ C × 2 ; 94 ◦ C × 30 , 61 ◦ C × 45 , 68 ◦ C × 1 /30 cycles; 68 ◦ C × 8 94 ◦ C × 2 ; 94 ◦ C × 30 , 61 ◦ C × 45 , 68 ◦ C × 1 /30 cycles; 68 ◦ C × 8 5 -GCAGAGTGGAGTTCCCAAAACC-3 5 -CCGCCTCTACTGGGTTGACTCCAAACTTCAC-3 ; 5 -GCTGACCTTTAGCCTGACGGTGGATG-3 5 -CCAAGGTCATTTGAGACTTTCGTCA-3 ; 5 -TGGTGCCATCTGCTGTTGTGT-3 5 -AGAGGACCACCCTGAGCAATGGCGG-3 ; 5 -GCGACCACGTTCCTCAGGTTGGGGATGAGG-3 5 -GGTGCTTTCTGCTAGGTCC-3 ; 5 -AGCAGCTTGGGCTTGTCCCAGA-3 Del, deletion; Dup, duplication; e, exon; F, forward primer; R, reverse primer; bp, base pair. LDLR by MLPA. DNA samples from 50 unrelated patients with a diagnosis of definite (N = 32) or possible (N = 18) heterozygous FH were analysed; of these, 14 were found to have a deletion or duplication of one or more exons in the LDLR. These data are summarised in Fig. 1 and Table 4, and points of interest described in more detail below. Panel A in Fig. 1 (Fig. 1A) shows the results from four patients in whom no major rearrangement was found. 3.1. Deletions of single exons DNA from two unrelated patients was found to have reduced copy number of exon 8 (Fig. 1B, samples 629 and 663); however, amplification and sequencing of exon 8 from genomic DNA revealed that both these individuals were heterozygous for a 4 bp duplication in this exon (bases GTGG1118–1121 , where base 1 is A of the ATG initiator codon) that encompassed the region of the exon where the Fig. 1. Results of MLPA analysis of the LDL-receptor gene (LDLR) in genomic DNA from FH patients. Peak heights for each PCR product were normalised relative to the control probes within each sample and then expressed relative to the mean peak height of each in at least five control DNA samples run at the same time. The x-axis represents the size of the PCR product from which the identity of the product is determined. (A) Representative MLPA patterns from FH patients in whom no rearrangements were detected. (B) Patients with small deletions of one or two exons at the 5 end of LDLR; ** indicates the peak obtained with the probe to SMARCA1, the control gene adjacent to the 5 end of the LDL receptor gene (see Fig. 3); ‡ indicates a control gene that is present at half copy number in two subjects. (C) patients with duplications in LDLR. (D) Patients with large deletions or deletions at the 3 end of LDLR; * indicates the peak obtained with the probe to ANKRD25, the control gene in the region flanking the 3 end of LDLR. Please cite this article in press as: Tosi I et al., Genetic defects causing familial hypercholesterolaemia: Identification of deletions and duplications in the LDL-receptor gene and summary of all mutations found in patients attending the Hammersmith Hospital Lipid Clinic, Atherosclerosis (2006), doi:10.1016/j.atherosclerosis.2006.10.003 ATH-9692; ARTICLE IN PRESS No. of Pages 10 4 I. Tosi et al. / Atherosclerosis xxx (2006) xxx–xxx Table 2 LDLR mutations identified in 153 unrelated heterozygous FH patients attending Hammersmith Hospital Lipid Clinic LDLR mutation Exon No. of index patients M-21L (nt A1 → T) Q12X (nt C97 → T) C25G (nt T136 → G)a C47X (nt C204 → A)a del nt GT196–197 C54W (nt T225 → G)a W66G (nt T260 → G) W66X (nt G261 → A) C68Y (nt G266 → A) D69G (nt A269 → C) E80K (nt G301 → A) del nt G303 nt C313+1 G → A nt C313+5 G → A C88Y (nt G326 → A E119D (nt G420 → C) C146X (C501 → A) Y167X (nt C564 → G) del G197 (nt GGT652−4 ) D200N (nt G661 → A) D200G (nt A662 → G) dup 21 nt663–683 D206E (nt C681 → G) del nt AC680−1 E207Q (nt G682 → C) E207X (nt G682 → T) C210X (nt C693 → A) ins C after nt C704 C227Y (nt G743 → A) S265R (nt C858 → A) D280A (nt A902 → C) D283N (nt G910 → A) D283E (nt C912 → G) D286H (nt G919 → C) C292Y (nt G938 → A) C292X (nt C939 → A) R329X (nt C1048 → T) R329P (nt C1048 → G) del nt C1008−31 ins GTGG at nt T1112 C356Y (nt G1130 → A) C358R (ntT1135 → C) Q363X/D365E (nt C1150 → T; C1158 → G) C371X (nt C1176 → A) nt C1216 A (splice) R385Q (nt G1217 → A) E387K (nt G1222 → A) Y398X (nt C1257 → G) V408M (nt G1285 → A) del nt G1358+55 , ins CGGCT Q434X (nt C1363 → T)a L458P (nt T1436 → C) D461H (nt G1444 → C) D461N (nt G1444 → A) del CT at (nt C1477−8 ) D471N (nt G1474 → A) V502M (nt G1567 → A) A519T (nt G1618 → A) Q540X (nt C1681 → T) G544A (nt G1694 → C) L578S (nt T1786 → C) ex 1 ex 2 ex 2 ex 2 ex 3 ex 3 ex 3 ex 3 ex 3 ex 3 ex 3 ex 3 intron 3 intron 3 ex 4 ex 4 ex 4 ex 4 ex 4 ex 4 ex 4 ex 4 ex 4 ex 4 ex 4 ex 4 ex 4 ex 5 ex 5 ex 6 ex 6 ex 6 ex 6 ex 6 ex 6 ex 6 ex 7 ex 7 ex 7 ex 8 ex 8 ex 8 ex 8 1 1 1 1 1 1 5 1 4 3 4 1 5 1 1 1 1 1 8 2 3 1 2 6 1 6 1 1 1 1 1 1 1 1 2 2 4 1 1 1 1 1 1 ex 8 ex 9 ex 9 ex 9 ex 9 ex 9 intron 9 ex 10 ex 10 ex 10 ex 10 ex 10 ex 10 ex 10 ex 11 ex 11 ex 11 ex 12 1 1 1 2 1 2 3 1 2 3 3 1 1 1 1 1 1 1 Table 2 (Continued ) LDLR mutation Exon No. of index patients P587R (nt C1828 → nt G1845+11 C → G) F598L (nt T1855 → C) W599R (nt T1858 → C) A606D (nt C1899 → A) R612C (nt C1897 → T) P628L (nt C1946 → T) del G1987 C660Y (nt C2042 → A) C656R (nt T2029 → C) P664L (nt C2054 → T) ins C at nt2061 A676P (nt G2089 → C)a R723Q (nt2231 → A) ex 12 intron 12 ex 13 ex 13 ex 13 ex 13 ex 13 ex 13 ex 14 ex 14 ex 14 ex 14 ex 14 ex 15 1 1 1 1 1 3 1 1 1 1 8 1 1 1 Deletion Deletion Deletion Deletiona Deletiona Deletion Deletion Deletion Deletion Deletion duplication duplication Deletion ex 1 ex 2–5 ex 2–6 ex 2–18 ex 1–18 ex 5 ex7 ex 7–8 ex 7–14 ex 13–14 ex 9–14 ex 11–12 ex 16–17 1 1 1 1 1 5 1 1 1 2 1 1 1 a Mutations not listed on LDLR mutation databases (http://www.ucl. ac.uk/fh/ and http://www.umd.necker.fr). probes annealed. Careful inspection of the MLPA results shows that the apparent copy number of exon 8 is more than half, perhaps suggesting that some amplification of the mutant allele occurred. DNA from two other patients was found to have half the normal copy number of exon 5 (Fig. 1B, samples 567 and 617); in both cases, deletion of exon 5 was confirmed by PCR amplification of genomic DNA across the deletion joint in the probands and in affected relatives. As shown in Fig. 2A for proband 567 and his hypercholesterolaemic daughter, amplification of genomic DNA with a forward primer located in exon 4 and a reverse primer in exon 6 produced the expected 2.2 kb normal product with DNA from the proband, his daughter and a control subject, together with an additional product of approximately 1.3 kb in the proband and his daughter. This mutation has been observed previously in the UK [6]. DNA from one patient (547 in Fig. 1B) had half the normal copy number of exon 1; the same deletion was found in DNA from the patient’s affected father by MLPA (data not shown). Sequencing of the promoter and exon 1 of the LDLR in amplified genomic DNA from the patient did not reveal any variation that could explain the apparent deletion of this exon, but the deletion joint could not be amplified with any of several primer sets tested. This is probably because intron 1 is very large and the deletion at the 5 end could have extended 27 kb upstream as far as the adjacent gene, SMARCA4 (Fig. 3) which was intact in the patient’s DNA (probe ** in Fig. 1B). Please cite this article in press as: Tosi I et al., Genetic defects causing familial hypercholesterolaemia: Identification of deletions and duplications in the LDL-receptor gene and summary of all mutations found in patients attending the Hammersmith Hospital Lipid Clinic, Atherosclerosis (2006), doi:10.1016/j.atherosclerosis.2006.10.003 ATH-9692; ARTICLE IN PRESS No. of Pages 10 I. Tosi et al. / Atherosclerosis xxx (2006) xxx–xxx 5 Table 3 LDLR mutations identified in 28 “homozygous” FH patients referred to Hammersmith Lipid Clinic or to Lipoprotein Group for genetic characterisation Gene Genotype LDLR LDLR LDLR LDLR LDLR LDLR LDLR LDLR LDLR LDLR LDLR LDLR LDLR LDLR LDLR LDLR LDLR LDLR LDLR LDLR LDLR LDLR LDLR LDLR AB AB AB AB AB AB AB AB ABb AB AB AB AB AB AAc AA AAc AA AA AA AAc AAc AAc AAc LDLRAP1f LDLRAP1 LDLRAP1 LDLRAP1 AB AAc AAc AA a b c d e f Mutation(s) Allele A Allele B L578S E80K C227Y P664L D69G D461N Del ex 13–15a C176Ra (nt T588 → C) P664Lb C68Y F220Sa (nt T722 → C) D280A E80K P664L P664L R385Pa (nt G1217 → C)d E387K C292X C292X Q540X W-18Xa (nt G11 → A) C281Wa (nt C906 → G) G528Da (nt G1646 → A) D112Na (nt G397 → A)d del ex 2–6 del exon 1–6a R329P Unknown D283E dup nt 663–683 R612C P664L P664Lb del ex 16–17 E207X S265R del nt A2292 a del G736 a – – – – – – – – – – UK UK UK UK UK UK UK UK UK UK UK UK/Greek (B) UK/Irish (B) Asian Indian Asian Indian Asian Indian Asian Indian Greek Cypriote Greek Cypriote AfroCaribbean Colombian Iraqi Kosovan Omani English Turkish (Lebanese) Pakistani Sardinian Mutation not found in heterozygous FH patients. Same mutation on different haplotype inherited from unrelated parents [21]. Parents known to be consanguineous. Mutations not described previously (see Table 2). Patients not known to be related, but common Greek mutation [25]. Formerly known as ARH, for autosomal recessive hypercholesterolaemia [26]. 3.2. Deletions of two or more exons As shown in Fig. 1B, DNA from patient 614 was found to half the normal copy number of exons 7 and 8 of the LDLR, Table 4 Deletions and rearrangements in LDLR detected by MLPA Patient DNA no. Major rearrangement Confirmation Affected relative PCR 567 659 617 549 580 564 565 629 547 614 560 397 556 Del ex 5 Del ex 1–18 Del ex 5 Dup ex 11–12 Del ex 13–14 Del ex 2–18 Del 2–6 Del ex 8 (4 bp del) Del ex 1 Del ex 7–8 Dup ex 9–14 Del ex 2–5 Del ex 16–17 Y Na Y Y N Y Y Y Y Y/N Y Y Y PCR – PCR PCR PCR – – DNA sequence – PCR PCR mRNA sequence PCR a Ethnic origin Observed in two independent samples of genomic DNA. and apparently also half the normal copy number of a control gene locus on chromosome 10p14 (probe ‡) which encodes a sequence 11 kb from the 10p telomere encoding the hypothetical gene LOC254312 [13]. Individual 614 was the sibling of the proband in our study, but the data revealed that the proband did not carry the deletion of exons 7 and 8, but had inherited the variant allele of chromosome 10 (Fig. 1B). No other DNA samples tested, including controls, were found to carry this variant, and we conclude that it occurs with an allele frequency of less than ∼0.005. Since this locus encodes a hypothetical gene for which there is no evidence that it might be associated with lipid metabolism, this gene variant was not investigated further. DNA from one patient (659 in Fig. 1D) was found to have half the normal copy number of exons 1–18, and since the entire coding region of LDLR was apparently deleted from one allele, this deletion would not be detected by Southern blotting with cDNA probes. No affected relatives were available from this subject, but the deletion was observed in two separate DNA samples from the same individual. The full extent of this deletion could not be determined by PCR, but Please cite this article in press as: Tosi I et al., Genetic defects causing familial hypercholesterolaemia: Identification of deletions and duplications in the LDL-receptor gene and summary of all mutations found in patients attending the Hammersmith Hospital Lipid Clinic, Atherosclerosis (2006), doi:10.1016/j.atherosclerosis.2006.10.003 ATH-9692; 6 No. of Pages 10 ARTICLE IN PRESS I. Tosi et al. / Atherosclerosis xxx (2006) xxx–xxx Fig. 2. Confirmation of deletions and duplications by PCR. (A) Genomic DNA from patient 567 (Pr), his daughter (Da) and a control subject (Co) was amplified with the indicated primers and the products analysed on a 1% agarose gel stained with ethidium bromide; the diagram above shows part of LDLR with the deleted exons shaded. (B) mRNA from immortalised B-lymphocytes from patient 397 (Pr) and a control subject (Co) was amplified by RT-PCR and sequenced with a primer from nt 1137–1087 in the cDNA, where 1 is the A of the ATG initiator codon; the sequence shown is the non-coding strand. (C) Genomic DNA from patient 580 (Pr) and a control subject (Co), was amplified and analysed as described in A above. (D) Amplified genomic DNA from patient 556 (Pr), his sibling (Si), father (Fa) and mother (Mo). (E) Amplified genomic DNA from patient 560 (Pr), her twin (Tw), another sibling (Si, patient 710 in Fig. 1) and a control subject (Co); Bl, water blank. (F) Amplified DNA from patient 549 (Pr) his uncle (Un, patient 713 in Fig. 1) and two control subjects (Co); Bl, water blank. both flanking control genes were present in apparently normal copy number (probes SMARCA4** and ANKRD25*, 33 kb downstream; see Fig. 3). DNA from another patient (564 in Fig. 1D) had half the normal copy number of exons 2–18; again the deletion joint could not be amplified but the downstream control gene (probe *) was present in normal copy number. Two further affected members of the same large fam- Fig. 3. Location of control genes adjacent to the LDL receptor. The location of the control genes was determined from the ENSEMBL website: http://www.ensembl.org/Homo sapiens/contigview?c=19:11082496.5; w=500000. ily severely affected by FH, the proband’s niece and a sibling, were found to have this same deletion by MLPA analysis. Again, this deletion was not detectable by Southern blotting with cDNA probes. DNA from patient 565 had half the normal copy number of exons 2–6 (Fig. 1D); this mutation has been observed previously in FH patients in the UK [20]. MLPA analysis showed that the affected mother of this patient carried the same deletion, but his son did not (data not shown). DNA from patient 397 had half the copy number of exons 2–5 (Fig. 1D) and her affected daughter was found to have the same deletion (data not shown). In an immortalised lymphocyte cell line from proband 397, the sequence of LDLR mRNA amplified by RT-PCR confirmed the deletion of exons 2–5 (Fig. 2B). DNA from one patient was found to have half the normal copy number of exons 13 and 14, and deletion of these two Please cite this article in press as: Tosi I et al., Genetic defects causing familial hypercholesterolaemia: Identification of deletions and duplications in the LDL-receptor gene and summary of all mutations found in patients attending the Hammersmith Hospital Lipid Clinic, Atherosclerosis (2006), doi:10.1016/j.atherosclerosis.2006.10.003 ATH-9692; No. of Pages 10 ARTICLE IN PRESS I. Tosi et al. / Atherosclerosis xxx (2006) xxx–xxx 7 Table 5 Genetic variation in PCSK9 in patient without known mutations Genetic variation in PCSK9 Number of individuals Location Sequence variant Hmz common Htz/Hmz rare Exon 1 Exon 1 Intron 4 Intron 4 Intron 5 Exon 9 Exon 9 15–16 ins L(+L) ins CTG at nt 287 R46L exon nt G381 → T Exon 4 nt C + 4 → T Exon 5 nt C − 7 → T Exon 5 nt A + 3 → G V460V nt G1624 → A I474V nt A1664 → G 20 23 31 22 24 29 26 5/0 1/1 1/0 10/0 8/0 3/0 6/0 exons from genomic DNA was confirmed by PCR across the deletion joint with a forward primer in exon 12 and a reverse primer in exon 15. As shown in Fig. 2C, the normal allele was not amplified (expected product of 6.3 kb) in DNA from either the proband or unaffected controls, but a 2.7 kb product was observed in DNA from the proband. Deletion of these two exons has also been observed previously in an FH patient in the UK population [27]. Finally, DNA from one patient was found to have half the normal copy number of exons 16 and 17, a deletion that was also confirmed by amplification across the deletion joint in genomic DNA from the patient and his mother. As shown in Fig. 2D, with PCR primers located in exons 15 (forward) and 18 (reverse), the expected normal allele of 8.6 kb could not be amplified with the conditions used, but a product of approx. 4.8 kb was obtained with DNA from the proband and his mother that was not observed with DNA from control subjects. 3.3. Duplications Two probands were found to have duplications of part of the LDLR. In one patient (549 in Fig. 1C), more than two copy numbers of exons 11 and 12 were present; this was also observed in his maternal uncle (713 in Fig. 1C). Amplification of genomic DNA with a forward primer in exon 12 and a reverse primer in exon 11 produced a product of approx. 2.0 kb with DNA from the proband and his uncle, but not with DNA from an unaffected control subject (Fig. 2F). This confirmed the presence of a tandem duplication of exons 11 and 12 in intron 12. Another patient (560 in Fig. 1C) was found to have multiple copies of exons 9–14, and this was also observed in her twin sister and a further sister (710 in Fig. 1C). Interestingly, in all three cases and in several separate MLPA runs, the copy number appeared to be greater than could be explained by a single duplication of these exons. As can be seen in Fig. 1C, all the duplicated peaks were present at approx. 3-fold the normalised level, compared with the expected 1.5-fold that is observed when there are three copy numbers (for example, see the duplication of exons 11 and 12 described above; DNA 713 and 549 in Fig. 1C). The presence of at least one tandem duplication of exons 9–14 was confirmed by PCR with a forward primer located in exon 14 and a reverse primer in exon 9; a product of approx. 2 kb was obtained with DNA from the proband, her affected twin and a second affected sister that was not obtained with DNA from a control individual (Fig. 2E). This confirmed the presence of a duplication of exons 9–14 in intron 14, but could not distinguish between one or more duplications of this gene segment. 3.4. Screening for mutations in APOB and PCSK9 Genomic DNA from 32/35 patients with no detectable mutation was screened for possible rare mutations in APOB and PCSK9; DNA was no longer available from the other three. For APOB, codons 3431–3584 (in exon 26) and 4310–4396 (in exon 29) were amplified and sequenced, but the only sequence variant found was the common polymorphism in codon N4311 [28]; 7 (22%) patients were heterozygous and 4 (12%) were homozygous for the less frequent G allele, while the remainder were homozygous for the more frequent A allele. For PCSK9, the coding exons and flanking sequences were sequenced; only known polymorphisms that have been detected in normolipaemic individuals were found [29], as shown in Table 5. 4. Discussion Out of 206 unrelated heterozygous FH patients who have now been analysed in detail, a genetic defect has been identified in 83% (171), as summarised in Table 6. These comprise 153 patients (74.2% of all FH patients) with 88 different point mutations and 13 different deletions or rearrangements in LDLR (i.e. major rearrangements comprise 13.6% of heterozygous LDLR mutations), 4 patients with two different mutations in PCSK9 (0.2% of all FH patients) [2] and 14 FDB patients (6.8% of all FH patients) [30]. In addition we have characterised 28 “homozygous” FH patients, 4 of whom have recessive hypercholesterolaemia due to mutations in LDLRAP1 (formerly known as ARH) [11,26], 13 are compound heterozygous FH patients of total or partial UK origin who have two different mutant LDLR alleles, one compound heterozygous FH patient of Asian Indian origin and 14 of non-UK origin who are all true homozygous FH with two identical LDLR alleles. All but two of the mutations found in the homozygous FH patients of UK origin have also been Please cite this article in press as: Tosi I et al., Genetic defects causing familial hypercholesterolaemia: Identification of deletions and duplications in the LDL-receptor gene and summary of all mutations found in patients attending the Hammersmith Hospital Lipid Clinic, Atherosclerosis (2006), doi:10.1016/j.atherosclerosis.2006.10.003 ATH-9692; No. of Pages 10 8 ARTICLE IN PRESS I. Tosi et al. / Atherosclerosis xxx (2006) xxx–xxx Table 6 Summary of genetic defects in FH patients attending Hammersmith Lipid Clinic Gene Type of mutation mutationsb No. of different mutations No. of unrelated HHa probands References Htz “Hmz” 153 24 [14–22] and unpublished data LDLR Point Rearrangementsc 85 13 PCSK9 Point mutationsb 2 4 0 [2] mutationsb 4 1 0 4 [11] ARH/LDLRAP1 Point Rearrangementsd APOB None a b c d ApoB3500 Definite FH Possible FH 14 [30] 18 17 HH, Hammersmith Hospital; htz, heterozygous; “hmz”, homozygous or compound heterozygous. Detected by sequencing of mRNA and/or DNA; details in Table 1. Detected by MLPA or Southern blotting. Detected by FISH. found in unrelated heterozygous patients, but many of the mutations in homozygous FH patients of non-UK origin are unique in this study. In addition, we have identified mutations in more than 200 affected relatives of 80 FH probands (data not shown) (Table 6). Analysis of DNA by MLPA was found to be a simple, rapid and robust means of detecting major rearrangements in the LDLR that compared favourably with other methods such as Southern blotting and long PCR, and some large deletions can only be detected by this method. In our experience, the quality of the DNA is critical for successful MLPA analysis and DNA prepared from blood by “quick” methods may not produce reliable results. Apparent deletions of single exons may be caused by polymorphisms or mutations involving one or a few base pairs in the exon, as we demonstrate here with the apparent deletion of exon 8. Therefore, for deletions of single exons the nucleotide sequence of the exon should always be determined, and the presence of a deletion confirmed, if possible, by PCR across the deletion joint. In the majority of cases in our study, the presence of a deletion or duplication in genomic DNA was confirmed by PCR across the deletion joint; the three exceptions were deletions of exon 1, 1–18 and 2–18, where it was impossible to predict suitable PCR primer sequences because there are no adjacent exons 5 to exon 1 or 3 to exon 18 known to be present, and the nearest flanking control genes were considered to be too distant. FISH can be used to confirm the presence of large deletions, but this requires access to fresh cells or an immortalised cell line [11]. In this study we have demonstrated the value of genetic diagnosis of FH in two critical cases where the clinical diagnosis was unclear. Of particular interest was the detection of deletion of exons 16 and 17, as the proband was a child who had had very severe hypercholesterolaemia and cutaneous xanthomatas from early infancy and was suspected of having homozygous FH. However, the diagnosis was unclear because the mother was reported as being normocholesterolaemic, although the father was reported as having hypercholesterolaemia caused by an LDLR mutation. A point mutation in exon 3 (C68Y), inherited from his father, had been identified elsewhere [31], but no further search for mutations had been performed. The patient’s father was subsequently referred to the Hammersmith Lipid Clinic where we carried out further analysis of the LDLR in several family members. Although the mother had been described as having normal plasma cholesterol levels (total plasma cholesterol 6.5 mmol/l), she was also found to carry the LDLR allele with the deletion; her relatively normal plasma cholesterol level can probably be explained by her diet, which results in her being extremely lean (body mass index <18 kg/m2 ). Thus we were able to confirm that the child was clearly compound heterozygous FH, having inherited a C68Y allele from the father and an allele with deletion of exons 16–17 from the mother, and should be treated accordingly. In the second case, the proband had been given a diagnosis of definite heterozygous FH on the basis of a raised plasma cholesterol value in the patient and a strong family history, including siblings with tendon xanthomas, hypercholesterolaemia and premature death due to coronary disease. We were able to confirm that although the affected sibling did indeed carry a mutation in LDLR, a deletion of exons 7 and 8, the proband did not carry this mutation. Although the proband had hypercholesterolaemia, it was less severe than that of the affected sibling (untreated plasma cholesterol 8.5 mmol/l versus 13.7 mmol/l) and was probably secondary to obesity and/or other environmental and genetic factors. Of the 35 patients in whom we failed to detect any mutations by MLPA analysis, we had also failed to detect an LDLR mutation by exon-by-exon sequencing of amplified genomic DNA. Complete sequencing of the exons and flanking sequences of PCSK9 failed to reveal any sequence variants that have not also been found in non-FH individuals [29], and no sequence variants were detected when regions of APOB encoding LDL-receptor binding domains [12] were sequenced in these patients. Of the patients with no known defect, almost half had a diagnosis of definite FH, implicating some inherited component to their disorder, although it is pos- Please cite this article in press as: Tosi I et al., Genetic defects causing familial hypercholesterolaemia: Identification of deletions and duplications in the LDL-receptor gene and summary of all mutations found in patients attending the Hammersmith Hospital Lipid Clinic, Atherosclerosis (2006), doi:10.1016/j.atherosclerosis.2006.10.003 ATH-9692; No. of Pages 10 ARTICLE IN PRESS I. Tosi et al. / Atherosclerosis xxx (2006) xxx–xxx sible that this may not be monogenic or may result from more complex interactions between gene variants and the environment. Nonetheless, we conclude that despite the recent addition of two new genes, namely LDLRAP1 and PCSK9, to the list of genes (LDLR and APOB) in which defects are known to be associated with severe inherited hypercholesterolaemia, there still appear to be a substantial number of patients with a clinical diagnosis of possible or even definite FH who have an unknown genetic defect. Acknowledgements Some of the patients in this study had previously been under the care of Professor Gilbert Thompson, or of Dr. Mary Seed at the Charing Cross Hospital, and we are indebted to them for their participation. Bruce Pottinger provided excellent technical assistance and MSc project students Mafalda Bourbon and Daniel Meechan identified some of the point mutations not previously published. Four of our index patients with LDLR rearrangements are part of the Simon Broome Register Study, and we are grateful to Prof. S. Humphries for sharing preliminary results. We are grateful to colleagues who sent us samples and entrusted to us the genetic diagnosis of their homozygous FH patients. References [1] Goldstein J, Hobbs H, Brown M. Familial hypercholesterolemia. In: Valle D, Scriver CR, Beaudet A, Sly WS, Childs B, Kinzler KW, Volgestein B, editors. The metabolic and molecular bases of inherited disease. New York: McGraw Hill; 2001. p. 2863–913. [2] Sun XM, Eden ER, Tosi I, et al. Evidence for effect of mutant PCSK9 on apolipoprotein B secretion as the cause of unusually severe dominant hypercholesterolaemia. Hum Mol Genet 2005;14:1161–9. [3] Top B, Koeleman BP, Gevers Leuven JA, et al. Rearrangements in the LDL receptor gene in Dutch familial hypercholesterolemic patients and the presence of a common 4 kb deletion. Atherosclerosis 1990;83:127–36. [4] Lelli N, Ghisellini M, Gualdi R, et al. Characterization of three mutations of the low density lipoprotein receptor gene in Italian patients with familial hypercholesterolemia. Arterioscler Thromb 1991;11: 234–43. [5] Aalto-Setala K, Koivisto UM, Miettinen TA, et al. Prevalence and geographical distribution of major LDL receptor gene rearrangements in Finland. J Intern Med 1992;231:227–34. [6] Sun XM, Webb JC, Gudnason V, et al. Characterization of deletions in the LDL receptor gene in patients with familial hypercholesterolemia in the United Kingdom. Arterioscler Thromb 1992;12: 762–70. [7] Garcia-Garcia AB, Real JT, Puig O, et al. Molecular genetics of familial hypercholesterolemia in Spain: ten novel LDLR mutations and population analysis. Hum Mutat 2001;18:458–9. [8] Schouten JP, McElgunn CJ, Waaijer R, et al. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res 2002;30:e57. [9] Scientific Steering Committee on behalf of the Simon Broome Register Group. Risk of fatal coronary heart disease in familial hypercholesterolaemia. BMJ 1991;303:893–6. 9 [10] Sun XM, Patel DD, Knight BL, et al. Comparison of the genetic defect with LDL-receptor activity in cultured cells from patients with a clinical diagnosis of heterozygous familial hypercholesterolemia. The Familial Hypercholesterolaemia Regression Study Group. Arterioscler Thromb Vasc Biol 1997;17:3092–101. [11] Eden ER, Patel DD, Sun XM, et al. Restoration of LDL receptor function in cells from patients with autosomal recessive hypercholesterolemia by retroviral expression of ARH1. J Clin Invest 2002;110:1695–702. [12] Fouchier SW, Kastelein JJ, Defesche JC. Update of the molecular basis of familial hypercholesterolemia in The Netherlands. Hum Mutat 2005;26:550–6. [13] MRC-Holland (http://www.mrc-holland.com). [14] Soutar AK, Knight BL, Patel DD. Identification of a point mutation in growth factor repeat C of the low density lipoprotein-receptor gene in a patient with homozygous familial hypercholesterolemia that affects ligand binding and intracellular movement of receptors. Proc Natl Acad Sci USA 1989;86:4166–70. [15] Webb JC, Sun XM, Patel DD, et al. Characterization of two new point mutations in the low density lipoprotein receptor genes of an English patient with homozygous familial hypercholesterolemia. J Lipid Res 1992;33:689–98. [16] Feher MD, Webb JC, Patel DD, et al. Cholesterol-lowering drug therapy in a patient with receptor-negative homozygous familial hypercholesterolaemia. Atherosclerosis 1993;103:171–80. [17] Gudnason V, King-Underwood L, Seed M, et al. Identification of recurrent and novel mutations in exon 4 of the LDL receptor gene in patients with familial hypercholesterolemia in the United Kingdom. Arterioscler Thromb 1993;13:56–63. [18] Sun XM, Patel DD, Bhatnagar D, et al. Characterization of a splicesite mutation in the gene for the LDL receptor associated with an unpredictably severe clinical phenotype in English patients with heterozygous FH. Arterioscler Thromb Vasc Biol 1995;15:219–27. [19] Webb JC, Patel DD, Shoulders CC, et al. Genetic variation at a splicing branch point in intron 9 of the low density lipoprotein (LDL)-receptor gene: a rare mutation that disrupts mRNA splicing in a patient with familial hypercholesterolaemia and a common polymorphism. Hum Mol Genet 1996;5:1325–31. [20] Webb JC, Sun XM, McCarthy SN, et al. Characterization of mutations in the low density lipoprotein (LDL)-receptor gene in patients with homozygous familial hypercholesterolemia, and frequency of these mutations in FH patients in the United Kingdom. J Lipid Res 1996;37:368–81. [21] Bourbon M, Fowler AM, Sun XM, et al. Inheritance of two different alleles of the low-density lipoprotein (LDL)-receptor gene carrying the recurrent Pro664Leu mutation in a patient with homozygous familial hypercholesterolaemia. Clin Genet 1999;56:225–31. [22] Naoumova RP, Neuwirth C, Pottinger B, et al. Genetic diagnosis of familial hypercholesterolaemia: a mutation and a rare non-pathogenic amino acid variant in the same family. Atherosclerosis 2004;174: 67–71. [23] Sun XM, Patel DD, Webb JC, et al. Familial hypercholesterolemia in China. Identification of mutations in the LDL-receptor gene that result in a receptor-negative phenotype. Arterioscler Thromb 1994;14: 85–94. [24] Pimstone SN, Sun XM, du Souich C, et al. Phenotypic variation in heterozygous familial hypercholesterolemia: a comparison of Chinese patients with the same or similar mutations in the LDL receptor gene in China or Canada. Arterioscler Thromb Vasc Biol 1998;18: 309–15. [25] Mavroidis N, Traeger-Synodinos J, Kanavakis E, et al. A high incidence of mutations in exon 6 of the low-density lipoprotein receptor gene in Greek familial hypercholesterolemia patients, including a novel mutation. Hum Mutat 1997;9:274–6. [26] Soutar AK, Naoumova RP, Traub LM. Genetics, clinical phenotype, and molecular cell biology of autosomal recessive hypercholesterolemia. Arterioscler Thromb Vasc Biol 2003;23:1963–70. Please cite this article in press as: Tosi I et al., Genetic defects causing familial hypercholesterolaemia: Identification of deletions and duplications in the LDL-receptor gene and summary of all mutations found in patients attending the Hammersmith Hospital Lipid Clinic, Atherosclerosis (2006), doi:10.1016/j.atherosclerosis.2006.10.003 ATH-9692; 10 No. of Pages 10 ARTICLE IN PRESS I. Tosi et al. / Atherosclerosis xxx (2006) xxx–xxx [27] Gudnason V, Muller DP, Lloyd JK, et al. Response to drugs and diet in a compound heterozygote for familial hypercholesterolaemia. Arch Dis Child 1995;73:538–40. [28] Navajas M, Laurent AM, Moreel JF, et al. Detection by denaturing gradient gel electrophoresis of a new polymorphism in the apolipoprotein B gene. Hum Genet 1990;86:91–3. [29] Kotowski IK, Pertsemlidis A, Luke A, et al. A spectrum of PCSK9 alleles contributes to plasma levels of low-density lipoprotein cholesterol. Am J Hum Genet 2006;78:410–22. [30] Myant NB. Familial defective apolipoprotein B-100: a review, including some comparisons with familial hypercholesterolaemia. Atherosclerosis 1993;104:1–18 [published erratum appears in Atherosclerosis 1994;105(2):253]. [31] Heath KE, Humphries SE, Middleton-Price H, et al. A molecular genetic service for diagnosing individuals with familial hypercholesterolaemia (FH) in the United Kingdom. Eur J Hum Genet 2001;9:244–52. Please cite this article in press as: Tosi I et al., Genetic defects causing familial hypercholesterolaemia: Identification of deletions and duplications in the LDL-receptor gene and summary of all mutations found in patients attending the Hammersmith Hospital Lipid Clinic, Atherosclerosis (2006), doi:10.1016/j.atherosclerosis.2006.10.003